Prevalence, characterisation and management of anthelmintic resistance in gastro-intestinal nematodes of Scottish sheep

The studies within this thesis have made a valuable contribution to our understanding of anthelmintic resistance in Scotland and in particular to the prevalence of benzimidazole (BZ) and ivermectin (IVM) resistance, the expression of multiple resistance and its management. Parasitic gastroenteritis (PGE) is a major welfare issue not only for Scottish, UK and European farmers but also for livestock producers throughout the world. Parasites such as Haemonchus, Trichostrongylus and Teladorsagia are estimated to cost the sheep industry hundreds of millions of dollars annually. To date control has largely been achieved using anthelmintics, but over reliance on anthelmintics has led to the development of multi class anthelmintic resistance (AR) and the realization that intensive chemoprophylaxis is not a sustainable approach for the control of nematodoses. The first two papers contributing to this thesis assessed the prevalence of benzimidazole (BZ) and ivermectin (IVM) resistance within ovine gastrointestinal nematode populations in Scotland. The prevalence of BZ resistance in selected Scottish lowland sheep farms was around 24% in 1991 but this had risen to over 80% by 2001. The first cases of ivermectin resistance in sheep were only detected in 2001 but a small scale survey in 2004 showed that 35% of the farms (6 from 17) surveyed had IVM resistance, with Teladorsagia and Trichostrongylus being identified as the resistant genera. The isolation of a triple class resistant T. circumcincta (MTci5) population has enabled research to focus on the important issue of the therapeutic and prophylactic management of this emerging problem. The third and fourth papers detail a series of controlled efficacy tests conducted on MTci5 that confirmed, in the short term at least, it should be possible to use a milbemycin (moxidectin; MOX) or combination treatments, with IVM and one other class of anthelmintic to control nematodoses (>90% efficacy) caused by adult and/or immature worms. However the study examining larval susceptibility highlighted the important role that immature stages can play in the selection and transmission of resistance. Currently there are no tests that can detect the presence of these resistant larval stages. The fifth paper outlines parasitological findings from the farm where MTci5 was isolated following the confirmation of multiple class resistance. Substantial efforts were made to find solutions to maintain sustainability and profitability of the enterprise though ultimately the use of MOX selected for a, predominately Teladorsagia, population against which the persistent activity of the compound was only negligible with the reappearance of eggs in faeces occurring between 21 and 28 days post treatment. Effective sustainable control of AR populations not only requires an understanding of the phenotypic and genotypic mechanisms that underpin resistance but also improved means of ensuring that our farmers are made aware of and utilize identified best practice approaches. The written and verbal responses of the farmers to questions relating to best practice advice (papers six and seven) would suggest that many of the recommendations for delaying the selection and transmission of AR (ACME, Moredun Foundation and sustainable control of parasites of sheep (SCOPS), DEFRA) are not being followed, recommendations such as the effective quarantine treatment of newly purchased animals and dosing animals at the manufacturers’ recommended dose rate were followed by only 20% and 56% of farmers respectively

2017 Scientific Consensus Statement: land use impacts on the Great Barrier Reef water quality and ecosystem condition, Chapter 2: sources of sediment, nutrients, pesticides and other pollutants to the Great Barrier Reef

This chapter provides an up-to-date review of the state of knowledge relating to the source of sediment and nutrients as well as pesticides and other pollutants delivered to the Great Barrier Reef from adjacent catchments. The strengths and limitations of the various datasets are also discussed. Collectively, sediment, nutrients, pesticides and other pollutants (e.g. petroleum hydrocarbons, pharmaceuticals) are described as ‘pollutants’. This chapter is focused on defining the major source areas of these pollutants across the Great Barrier Reef, how these sources have varied in space and time, the major processes (e.g. hillslope, gully and streambank erosion) delivering these pollutants, their relative loads to the Great Barrier Reef and a summary of the main drivers in terms of land use, land condition and agricultural practices. Plot- and paddock-scale studies, including the effectiveness of remediation approaches, are summarised in Chapter 4. Acknowledging that all forms of data used to estimate pollutant loads to the Great Barrier Reef have constraints and limitations, this review uses a ‘multiple lines of evidence’ approach and draws on data from three main sources. These include the Queensland Government load monitoring data, the latest Queensland Government whole of Great Barrier Reef Source Catchments modelling results (which underpin the Report Card 2015) as well as a summary of the numerous individual research projects and synthesis reports published over the last four years. Data and information are included that was published, publicly available and that had undergone a peer review process. In a few cases, grey literature (e.g. consulting reports) and journal publications currently in review are included. A synthesis of the broad findings of this chapter are outlined below and in Table 1. A detailed description of what has changed since the last Scientific Consensus Statement is provided in Table 20

Assessment of the relative risk of water quality to ecosystems of the Great Barrier Reef. A report to the Department of the Environment and Heritage Protection, Queensland Government, Brisbane - Report 13/28

A risk assessment method was developed and applied to the Great Barrier Reef (GBR) to provide robust and scientifically defensible information for policy makers and catchment managers on the key land-based pollutants of greatest risk to the health of the two main GBR ecosystems (coral reefs and seagrass beds). This information was used to inform management prioritisation for Reef Rescue 2 and Reef Plan 3. The risk assessment method needed to take account of the fact that catchment-associated risk will vary with distance from the river mouth, with coastal habitats nearest to river mouths most impacted by poor marine water quality. The main water quality pollutants of concern for the GBR are enhanced levels of suspended sediments, excess nutrients and pesticides added to the GBR lagoon from the adjacent catchments. Until recently, there has been insufficient knowledge about the relative exposure to and effects of these pollutants to guide effective prioritisation of the management of their sources

Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Prevalence, characterisation and management of anthelmintic resistance in gastro-intestinal nematodes of Scottish sheep

The studies within this thesis have made a valuable contribution to our understanding of anthelmintic resistance in Scotland and in particular to the prevalence of benzimidazole (BZ) and ivermectin (IVM) resistance, the expression of multiple resistance and its management. Parasitic gastroenteritis (PGE) is a major welfare issue not only for Scottish, UK and European farmers but also for livestock producers throughout the world. Parasites such as Haemonchus, Trichostrongylus and Teladorsagia are estimated to cost the sheep industry hundreds of millions of dollars annually. To date control has largely been achieved using anthelmintics, but over reliance on anthelmintics has led to the development of multi class anthelmintic resistance (AR) and the realization that intensive chemoprophylaxis is not a sustainable approach for the control of nematodoses. The first two papers contributing to this thesis assessed the prevalence of benzimidazole (BZ) and ivermectin (IVM) resistance within ovine gastrointestinal nematode populations in Scotland. The prevalence of BZ resistance in selected Scottish lowland sheep farms was around 24% in 1991 but this had risen to over 80% by 2001. The first cases of ivermectin resistance in sheep were only detected in 2001 but a small scale survey in 2004 showed that 35% of the farms (6 from 17) surveyed had IVM resistance, with Teladorsagia and Trichostrongylus being identified as the resistant genera. The isolation of a triple class resistant T. circumcincta (MTci5) population has enabled research to focus on the important issue of the therapeutic and prophylactic management of this emerging problem. The third and fourth papers detail a series of controlled efficacy tests conducted on MTci5 that confirmed, in the short term at least, it should be possible to use a milbemycin (moxidectin; MOX) or combination treatments, with IVM and one other class of anthelmintic to control nematodoses (>90% efficacy) caused by adult and/or immature worms. However the study examining larval susceptibility highlighted the important role that immature stages can play in the selection and transmission of resistance. Currently there are no tests that can detect the presence of these resistant larval stages. The fifth paper outlines parasitological findings from the farm where MTci5 was isolated following the confirmation of multiple class resistance. Substantial efforts were made to find solutions to maintain sustainability and profitability of the enterprise though ultimately the use of MOX selected for a, predominately Teladorsagia, population against which the persistent activity of the compound was only negligible with the reappearance of eggs in faeces occurring between 21 and 28 days post treatment. Effective sustainable control of AR populations not only requires an understanding of the phenotypic and genotypic mechanisms that underpin resistance but also improved means of ensuring that our farmers are made aware of and utilize identified best practice approaches. The written and verbal responses of the farmers to questions relating to best practice advice (papers six and seven) would suggest that many of the recommendations for delaying the selection and transmission of AR (ACME, Moredun Foundation and sustainable control of parasites of sheep (SCOPS), DEFRA) are not being followed, recommendations such as the effective quarantine treatment of newly purchased animals and dosing animals at the manufacturers’ recommended dose rate were followed by only 20% and 56% of farmers respectively.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

2017 Scientific Consensus Statement: land use impacts on the Great Barrier Reef water quality and ecosystem condition, Chapter 2: sources of sediment, nutrients, pesticides and other pollutants to the Great Barrier Reef

This chapter provides an up-to-date review of the state of knowledge relating to the source of sediment and nutrients as well as pesticides and other pollutants delivered to the Great Barrier Reef from adjacent catchments. The strengths and limitations of the various datasets are also discussed. Collectively, sediment, nutrients, pesticides and other pollutants (e.g. petroleum hydrocarbons, pharmaceuticals) are described as ‘pollutants’. This chapter is focused on defining the major source areas of these pollutants across the Great Barrier Reef, how these sources have varied in space and time, the major processes (e.g. hillslope, gully and streambank erosion) delivering these pollutants, their relative loads to the Great Barrier Reef and a summary of the main drivers in terms of land use, land condition and agricultural practices. Plot- and paddock-scale studies, including the effectiveness of remediation approaches, are summarised in Chapter 4. Acknowledging that all forms of data used to estimate pollutant loads to the Great Barrier Reef have constraints and limitations, this review uses a ‘multiple lines of evidence’ approach and draws on data from three main sources. These include the Queensland Government load monitoring data, the latest Queensland Government whole of Great Barrier Reef Source Catchments modelling results (which underpin the Report Card 2015) as well as a summary of the numerous individual research projects and synthesis reports published over the last four years. Data and information are included that was published, publicly available and that had undergone a peer review process. In a few cases, grey literature (e.g. consulting reports) and journal publications currently in review are included. A synthesis of the broad findings of this chapter are outlined below and in Table 1. A detailed description of what has changed since the last Scientific Consensus Statement is provided in Table 20

Backcrossing approach to introgress ivermectin resistance-conferring genes from resistance strains into the MHco3(ISE) genetic background.

<p>Diagrammatic representation of overall backcrossing scheme (A). Schematic representation of experimental aim and summary of nomenclature used (B). Genome of the MHco4(WRS) ivermectin resistant strain represented in red and the genome of the MHco3(ISE) susceptible reference strain represented in blue.</p

Visualisation of genetic differentiation between <i>H. contortus</i> parental and backcross strains.

<p>Pairwise F_ST estimates and principal component analysis of parental and 4^th generation backcross isolates pre and post ivermectin treatment (0.1 mg/kg ivermectin) using multi-locus genotype data. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002534#ppat-1002534-g003" target="_blank">Figure 3A, B and C</a> shows the data for the MHco3/4 cross and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002534#ppat-1002534-g003" target="_blank">Figure 3D, E and F</a> for the MHco3/10 cross. Both F_ST and PCA analysis was performed with 9 loci and also with 8 loci (when Hcms8a20 was excluded from this latter analysis, since it shows evidence of genetic linkage to the ivermectin resistance phenotype). Figure A and D show pairwise F_ST estimates: values in the tables below the diagonal are for analysis of all 9 loci and above the diagonal for the 8 loci (excluding Hcms8a20). Genetic differentiation between isolates at significance level, p<0.01 (highlighted in bold and italics). Asterisk indicates level of genetic differentiation is marginal (significance at p<0.05). PCA analysis for all 8 loci are shown in panels B and E and for the 9 loci (including 8a20) in panels C and F. Each data point represents a single worm based on a multi-locus genotype of 9 or 8 markers.</p